DESCRIPTION: The goal of this grant is to develop a new strategy for joining physiological function to biochemical behavior in proteins. This approach combines protein structure, mechanism, and engineering with evolutionary analyses. Central to this analysis is the reconstruction of evolutionary history of the protein superfamily from sequence data, and preparing ancient proteins from extinct organisms in the laboratory where they can be studied. The evolution of molecular and biological behavior is set in a historical context by correlating it with the evolution of organisms (deduced from paleontology) and the surrounding ecology, allowing construction of testable hypotheses concerning the structure-behavior relationships in this family, mechanisms by which these compounds exert their biological activities, and possible physiological function(s) of the proteins, as well as allowing to engineer new proteins with desired behaviors. The work here will use ribonuclease as a system to develop this strategy. Bovine pancreatic RNase A belongs to a superfamily of proteins that has, in various members, evolved to an enormous range of interesting biological behaviors. RNase homologs are known that are immunosuppressive, block the growth of tumor cells (But not normal cells), kill Schistosoma and Trichinella, cause neurological damage, cause lung damage in asthmatic patients, display lectin-like behavior, inhibit infection of mammalian cells by HIV-1, or do none of these. These behaviors have medical relevance; several RNase variants are now in clinical and preclinical stages of testing for their useful biomedical activities. In the next phase of this ongoing research program, the physiological significance of biological behaviors of seminal RNase will be assessed, a crystal structure solved to determine the mechanism by which seminal RNase binds and melts duplex DNA, the impact of the introduction of cysteines on the kinetics of folding and the thermodynamics of the folded structure will be examined and new sequence data will be collected to complete the model of the evolutionary history of this important class of molecules. This study will show how new biomolecular function is created in higher organisms, by mutation, insertion, deletion gene duplication and recruitment of duplicate genes. This evolutionary approach differs from that pursued in other laboratories, and this work will continue to develop a new paradigm where evolutionary information is integrated with chemical and biological information to solve biomedical research problems in a "post-genome" environment.